Method for automatic online detection of at least one biological target substance in a liquid and online analyzer

Abstract

A method for automated online detection of at least one biological target substance in a liquid using an online analyzer includes: transporting a first volume of liquid into a reaction chamber; concentrating biomolecules in the first volume of liquid by binding to a plurality of particles in the reaction chamber; transporting the particles with the bound biomolecules into a detection unit having a first microfluidics unit and a second microfluidics unit fluidically connectable thereto; releasing and/or isolating nucleic acids from the biomolecules bound on the particles; transporting an eluate of the released and/or isolated nucleic acids into the second microfluidics unit; amplifying a target nucleic acid in the second microfluidics unit; registering a measurement signal representing progress of the amplifying and/or on a number of copies of the target nucleic acid, and determining of the target substance in the sample based on the registered measurement signal.

1 - 18 . (canceled) 
     
     
         19 . A method for automated online detection of at least one biological target substance in a liquid using an online analyzer, the method comprising:
 transporting a predetermined first volume of the liquid via a sample supply line into a reaction chamber of a process unit of the online analyzer;   concentrating biomolecules contained in the first volume of liquid by binding to a plurality of particles in the reaction chamber;   transporting the particles with the bound biomolecules into a detection unit of the online analyzer, wherein the detection unit includes a first microfluidics unit and a second microfluidics unit fluidically connectable with the first microfluidics unit;   releasing and/or isolating nucleic acids from the biomolecules bound on the particles;   transporting an eluate comprising the released and/or isolated nucleic acids into the second microfluidics unit;   amplifying at least one target nucleic acid of the eluate in the second microfluidics unit;   registering a measurement signal, which represents a measured variable dependent on progress of the amplifying and/or on a number of copies of the at least one target nucleic acid in the second microfluidics unit; and   qualitatively and/or quantitatively determining the biological target substance in the sample based on the registered measurement signal using a control electronics of the analyzer.   
     
     
         20 . The method according to  claim 19 , wherein, after the concentrating of biomolecules contained in the first volume of liquid, the first volume of liquid is removed from the reaction chamber, a second volume of the liquid is introduced into the reaction chamber, and biomolecules contained in the second volume of liquid are concentrated in the reaction chamber by binding to the particles remaining in the reaction chamber. 
     
     
         21 . The method according to  claim 19 , wherein the particles are magnetic or paramagnetic particles, to which the biomolecules bind unselectively. 
     
     
         22 . The method according to  claim 19 , wherein the concentrating of biomolecules contained in the liquid includes introducing an alginate solution and a salt of a divalent or polyvalent cation, or of an acid, into the reaction chamber, wherein an alginate gel-biomolecule complex forms on the particles. 
     
     
         23 . The method according to  claim 21 , wherein the transporting of the particles with the bound biomolecules into the detection unit comprises:
 transporting the particles using at least one of: a magnet movable relative to the reaction chamber; a pin; a magnetic pin; and a pipetting apparatus, into a region of the reaction chamber where a fluid line connects the reaction chamber fluidically with the first microfluidics unit; and   rinsing the particles through the fluid line into the first microfluidics unit.   
     
     
         24 . The method according to  claim 19 , wherein the releasing and/or isolating of nucleic acids from the biomolecules bound on the particles in the first microfluidics unit comprises:
 contacting the particles with the bound biomolecules with one or more lysis reagents so as to release nucleic acids of the adsorbed biomolecules;   binding the released nucleic acids on the particles; and   washing the particles with the bound nucleic acids with a wash solution.   
     
     
         25 . The method according to  claim 24 , wherein the method further comprises:
 releasing the nucleic acids from the particles by eluting; and   transporting an eluate comprising the nucleic acids into the second microfluidics unit for subsequent amplification.   
     
     
         26 . The method according to  claim 19 , wherein the releasing and/or isolating of nucleic acids from the biomolecules bound on the particles in the first microfluidics unit comprises:
 thermal releasing of the biomolecules bound on the particles, or of nucleic acids contained in the biomolecules, in one of water, a tris buffer, or a buffer solution, which buffer solution comprises a phosphate-buffered, salt solution; and   transporting the buffer solution with the biomolecules or nucleic acid dissolved therein into the second microfluidics unit for subsequent amplification.   
     
     
         27 . The method according to  claim 19 , wherein the control electronics of the online analyzer executes all method steps automatically. 
     
     
         28 . An online analyzer for detection of at least one biological target substance in a liquid according to the method of  claim 19 , the online analyzer comprising:
 a control electronics; a process unit; and a detection unit,   wherein the process unit includes at least one reaction chamber and a sample supply line connected with the at least one reaction chamber, and wherein the process unit is adapted to receive a predetermined volume of the liquid in the at least one reaction chamber and to concentrate biomolecules contained in the liquid by binding to particles,   wherein the detection unit includes:
 fluidically connectable with the reaction chamber, a first microfluidics unit, which is adapted to release and/or to isolate nucleic acids from the biomolecules bound on the particles; 
 connected with the first microfluidics unit, a second microfluidics unit, which is adapted to receive an eluate comprising the released and/or isolated nucleic acids and to amplify a target nucleic acid; and 
 a sensor adapted to produce a measurement signal dependent on progress of an amplification performed in the second microfluidics unit and/or on a number of copies of the target nucleic acid in the second microfluidics unit and to output said measurement signal to the control electronics, and 
   wherein the control electronics is adapted to determine the biological target substance in the liquid, qualitatively and/or quantitatively, based on the measurement signal output from the sensor.   
     
     
         29 . The online analyzer according to  claim 28 , further comprising a sample pump, wherein the control electronics is adapted to control the sample pump for transporting a predetermined volume of liquid into the reaction chamber. 
     
     
         30 . The online analyzer according to  claim 28 , further comprising:
 at least one reaction component reservoir, which contains a reaction component for the concentrating of the biomolecules in the reaction chamber, wherein the reaction component comprises at least one of: an alginate solution, a salt of a divalent or polyvalent cation, a salt of an acid, and magnetic or paramagnetic particles; and   a reaction components supply line connecting the at least one reaction component reservoir with the reaction chamber, wherein the control electronics is adapted to dose, or meter, a predeterminable amount of the reaction component into the reaction chamber.   
     
     
         31 . The online analyzer according to  claim 28 , further comprising means for stirring the sample contained in the reaction chamber. 
     
     
         32 . The online analyzer according to  claim 28 , further comprising a magnet, which is so orientable relative to the reaction chamber via the control electronics that magnetic or paramagnetic particles contained in the reaction chamber are transported by a magnetic force of the magnet into a region of the reaction chamber, in which region a fluid line attached to the reaction chamber connects the reaction chamber fluidically with the first microfluidics unit. 
     
     
         33 . The online analyzer according to  claim 28 , wherein the first microfluidics unit and the second microfluidics unit are disposed together in a replaceable cartridge. 
     
     
         34 . The online analyzer according to  claim 28 , wherein the first microfluidics unit is adapted to receive the particles with bound biomolecules and to contact the particles with one or more lysis reagents so as to release nucleic acid of the adsorbed biomolecules, then to bind the released nucleic acids to the particles, and to wash the particles with the bound nucleic acids one or more times with a wash solution, and
 wherein the control electronics is adapted to control a transport of the particles and reagents through the first microfluidics unit.   
     
     
         35 . The online analyzer according to  claim 34 , wherein the first microfluidics unit is adapted further to remove the nucleic acids from the particles by elution and to transport the eluate into the second microfluidics unit via a fluid line connecting the first microfluidics unit with the second microfluidics unit, and
 wherein the control electronics is adapted to control the elution and the transport of the eluate.   
     
     
         36 . The online analyzer according to  claim 28 , wherein the first microfluidics unit is configured to receive the particles with bound biomolecules and to thermally release the biomolecules bound to the particles, or release the nucleic acids contained in the biomolecules, in water, in a tris buffer, or a buffer solution, which includes a phosphate-buffered, salt solution; and
 wherein the control electronics is configured to control the thermal releasing of the biomolecules and to transport the buffer solution with the biomolecules, or nucleic acids, dissolved therein into the second microfluidics unit for subsequent amplification.   
     
     
         37 . The online analyzer according to  claim 36 , wherein the tris buffer includes a complexing reagent. 
     
     
         38 . The online analyzer according to  claim 37 , wherein the complexing reagent is selected for complexing divalent or polyvalent cations bound on the particles with the biomolecules.